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J Integr Plant Biol.

• Research Article • Previous Articles    

Enhancing genetic transformation efficiency in cucurbit crops through AtGRF5 overexpression: Mechanistic insights and applications

Yang Li1†, Naonao Wang1†, Jing Feng1†, Yue Liu1†, Huihui Wang1, Shijun Deng1, Wenjing Dong1, Xiaofeng Liu1, Bingsheng Lv1, Jinjing Sun2, Kuipeng Xu1, Huimin Zhang1, Zhonghua Zhang1* and Sen Chai1*   

  1. 1. Engineering Laboratory of Genetic Improvement of Horticultural Crops of Shandong Province, College of Horticulture, Qingdao Agricultural University, Qingdao 266109, China
    2. State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China

    †These authors contribute equally to this work.
    *Correspondences: Zhonghua Zhang (zhangzhonghua@qau.edu.cn); Sen Chai (chaisen@qau.edu.cn, Dr. Chai is fully responsible for the distributions of all materials associated with this article)
  • Received:2024-09-29 Accepted:2025-03-17 Online:2025-04-11
  • Supported by:
    This work was supported by the National Natural Science Foundation of China (32225044 to Z.Z., 32130093 to Z.Z., 32102404 to S.C.), and the “Taishan Scholar” Foundation of the People's Government of Shandong Province.

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Abstract: Transgenic and gene-editing technologies are essential for gene functional analysis and crop improvement. However, the pleiotropic effects and unknown mechanisms of morphogenic genes have hindered their broader application. In this study, we employed the one-step de novo shoot organogenesis (DNSO) method, and demonstrated that overexpression of the morphogenic gene Arabidopsis thanalia GROWTH-REGULATING FACTOR 5 (AtGRF5) significantly enhanced genetic transformation efficiency in cucurbit crops by promoting callus proliferation and increasing dense cells during regeneration. High-resolution time-series transcriptomics and single-cell RNA sequencing revealed that AtGRF5 overexpression induced auxin-related genes and expanded stem cell populations during cucumber DNSO. Using DNA-affinity purification sequencing (DAP-seq) in combination with spatiotemporal differential gene expression analysis, we identified CsIAA19 as a key downstream target of AtGRF5, with its modulation playing a pivotal role in regeneration. Rescuing CsIAA19 in AtGRF5-overexpressing explant reversed the enhanced callus proliferation and regeneration. To address growth defects caused by AtGRF5 overexpression, we developed an abscisic acid-inducible AtGRF5 expression system, significantly improving transformation and gene-editing efficiency across diverse genotypes while minimizing pleiotropic effects. In summary, this research provides mechanistic insights into AtGRF5-mediated transformation and offers a practical solution to overcome challenges in cucurbit crop genetic modification.

Key words: auxin, cucurbit crops, genetic transformation, GRF, regeneration, scRNA‐Seq

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